This work was submitted to and approved by the Regional Ethic Committee and registered to the French Ministère de la Recherche. For muscle injuries, 8-12 weeks old male C57BL/6 mice (Janvier) were anesthetized with isoflurane and 80µL of 10 µM cardiotoxin (Sigma, C9759) or 50% glycerol in saline solution (NaCl 0.9%) were injected into the right quadriceps. For ScAT grafting, small pieces of ScAT (10mg) from Tg(Cd34-EGFP)MF6Gsat/Mmcd referred to as CD34-GFP mice (MMRRC) were grafted into ScAT of WT C57BL/6 mice by surgically skin incision, and after 7 days grafted mice were injured. For platelet depletion, mice were injected intraperitoneally with platelet depleting antibody (anti-GPIb, EMFRET) in saline solution (2mg/kg). Blood was collected under anesthesia in inferior cava vein with G25 needle and 1mL syringe coated with PBS/Heparin (20U/mL), and then samples were stored at 4°C to perform plasma or platelets isolation. Quadriceps muscle, sub cutaneous (Sc) and perigonadic (PG) adipose tissues (AT) were harvested for cell isolation. Liver, heart, kidney and front limb were dissected and fresh frozen at -80°C for genomic DNA or RNAs extraction. For lipectomy model, animals were anesthetized with isoflurane and a skin incision was performed above ScAT lymph node. ScAT was then removed using forceps to disrupt conjunctive tissue adherences, and blood vessels located at the extremities were cauterized. Wound was closed using surgical clips, and animals were monitored daily for 5 to 7 days before removal of the clips. Muscle injured results were compared to non-injured animals, and lipectomy results were compared to sham (skin incision only) animals.
Human Clinical Study.
Young active men (age 23.3 ± 1.7 years; BMI 23.3 ± 0.4 kg.m-²; VO2max 47.5 ± 1.6 ml.kg-1.min-1) were recruited to partake in this study. The protocol was approved by the Dublin City University Ethics Committee and all subjects gave written informed consent. Participants were instructed to refrain from exercise and to replicate food intake the day before each trial. In the morning, following an overnight fast, participants lay on a bed for 1-hr after arriving at the lab. A blood sample was taken and they then exercised on a bicycle ergometer at 60% VO2 peak for 1-hr. VO2 peak was determined on a cycle ergometer starting at 70 watts and increasing in 30-watt increments every 3 minutes until exhaustion. A blood sample was taken at the end of the exercise. The intensity was determined using the results of an incremental exercise test to exhaustion. GDF15 protein levels in blood samples were determined by ELISA (R&D Systems).
Human adipose tissue samples.
AT was obtained from patients who provided prior written informed consent according to the ethics committees of Toulouse Hospitals. AT was harvested during plastic surgery (abdominoplasty) from 3 adult patients (female, age 47.6 ± 3.7 years, BMI 26.7 ± 0.9 kg.m-²) at Rangueil Hospital (part of CHU of Toulouse).
Human AT-SVF isolation.
Human adipose tissue was digested for 45 minutes in α-MEM (GIBCO) containing collagenase (NB 4 , Coger, 0.4 IU/mL), Dispase II (Roche, 1.6 UI/mL, Basel), in a water-shaking bath at 37°C. After digestion, an equal volume of α-MEM was added to stop enzymatic digestion. The cells were passed through a 100 μm filter (Steriflip, Millipore, Billerica, MA) and then centrifuged. The pellet was resuspended in α-MEM containing 2% PLP and the total number of cells in the SVF was counted.
Murine AT- or muscle- SVF isolation.
Freshly harvested tissues were minced and stroma vascular fractions (SVF) were obtained by enzymatic digestion. PGAT and ScAT were digested with collagenase (NB4, Coger; 0,4 U/mL) and DNAse (1%, Roche) in αMEM (GIBCO) at 37°C for 45 and 60 min respectively under constant agitation. After centrifugation (300g, 10min, RT) and elimination of the floating adipocytes and the supernatant, the pellet containing the SVF was resuspended in erythrocyte lysis buffer (155 mmol/L NH4Cl; 5,7 mmol/L K2HPO4; 0,1 mmol/L EDTA, pH 7.3). After filtration through 34µm sieve and centrifugation (300g, 10min, RT), cells were resuspended in autoMACS® Running Buffer (Miltenyi Biotec). Quadriceps muscles were digested with collagenase B (0,5 U/mL, Roche) and dispase II (2,4 U/mL; Roche) in Hank’s Balanced Saline Solution (HBSS)+2,5 mM Ca2+ for 2 rounds of 30 min at 37°C under agitation separated by mechanical dissociation through G18 syringe. Reaction was stopped by adding a 2 volumes of αMEM + 10%NCS, then samples were filtered through 34µm sieve and centrifuged (300g, 10 min, RT) to eliminate supernatant and to resuspend the pellets in autoMACS® Running Buffer.
ASCs, FAPs, Platelets isolation.
For ASCs sorting, ScAT-derived SVF was depleted in CD45+ and CD31+ cells using anti-CD45-FITC and anti-CD31-FITC microbeads and autoMACS® Pro Separator (MACS Cell Separation, Miltenyi Biotec SAS) according to the manufacturer’s instructions. ASCs were plated at a density of 80,000 cells/cm2 in αMEM + 10% NCS for 3 days with 5% CO2 for further co-incubation with platelets.
For FAPs sorting, muscle derived SVF cells were stained with anti-CD45-PE, anti-CD31-PE and anti-Sca1-BV421 for 20 min and sorted with FACS ARIA (BD Biosciences). Sorted cells were stored at -20°C for further DNA extraction.
For platelets isolation, blood was centrifuged (5 min,500g, RT), and the plasma rich platelets (PRP) was resuspended in Tyrode’s Buffer. Samples were centrifuged for 2 rounds (1900g, 8 min) and platelets were stained with PKH26 for 5min at RT°. After another centrifugation step (1900, 8 min, twice), platelets were resuspended in αMEM + 10%NCS at 106 platelets/mL.
Plated ASCs were incubated with blocking antibody anti-Podoplanin (10µg/mL) before being co-cultured with 100.000 platelets for 1 hour (37°C, 5%CO2). Then, medium was removed, cells were rinsed with phosphate-buffered saline (PBS) and fixed with 3,7% PFA. Nuclei were stained with DAPI. Acquisition and analysis of data were performed with Operetta® system (Perkin Elmer).
For adipogenic differentiation, SVF cells were plated at a density of 60 000 cells/cm² in αMEM + 10%NCS at 37°C with 5% CO2. After 24 hours, medium was removed to eliminate the non-adherent cells, and replaced by adipogenic medium (αMEM, 2% NCS, Dexamethazone 33nM, Insulin 5mg/mL, Rosiglitazone 1µM, T3 10µM, Transferrin 10µg/mL). After 3 days of incubation, medium was removed and cells were frozen at -20°C for RNAs extraction.
Cell Migration Assay.
ASC migration assay was performed using the IncuCyte® S3 Live-Cell Analysis System (Essenbioscience). ASCs were isolated from mice ScAT or human samples as described above and were plated at a density of 2000 cell per well in a 96 well plate including a reservoir and an optically clear membrane insert assembly with laser-etched 8µm pore. For mice migration assay, the reservoirs were loaded with 200 µl of plasma (50%) obtained from animals injured or not. For human migration assay, the reservoirs were loaded with 200 µl of human serum at 25 % from healthy volunteers before or 1 hour after exercise. Cell migration across the pores was automatically quantified for over 30h or 40h for human and mouse ASCs respectively. Migration was analyzed via the Incucyte S3 software (Essenbioscience) and area under curves (AUC) were calculated and compared using GraphPad Prism software (GraphPad Software).
Flow Cytometry Analysis.
Isolated cells from ATs or BM were incubated (25 minutes, 4°C, dark) with Phycoerythrin (PE) CD31, CD45 or α7int, Allophycocyanin (APC)-Podoplanin or CD140a, Fluorescein isothiocyanate (FITC)-CD31, CD45 or Sca1, Sca-1-V500 (BD Bio-sciences), Brilliant violet (BV) 421-CXCR4 or Sca1 antibodies or the appropriate isotype controls. After washing, the labeled cells were quantified on LSR Fortessa flow cytometer and analyzed using FACSDiva software (BD Biosciences).
Proliferation in vivo was assessed with the Click Plus EdU 488 Flow Kit (Life Technologies) following the manufacturer’s instructions. Briefly, animals were injected IP with Edu (40 mg/g) just after the muscle injury, (i.e. 24 hr before sacrifice, such as described by Lemos et al. 9). Isolated muscle SVF-derived cells were fixed and permeabilized and further incubated for 30min with a Click It reaction cocktail to reveal Edu staining with Alexa Fluor 488 in proliferative cells.
For apoptose/necrose evaluation, In Situ Cell Death Detection Kit (ROCHE) was used following manufacturer’s instructions. Shortly, isolated SVF-derived cells were fixed and permeabilized before the TUNEL staining (labelling solution + enzyme) for 1 hour at 37°C. Stained cells were quantified by flow cytometry on LSR Fortessa (BD Biosciences).
RNAs and genomic DNA extraction, and RT-qPCR.
Extraction was realized on frozen cells or whole tissues using RNA Extractions Mini kit (QiaGEN) following manufacturer’s instructions. Briefly, samples were unfrozen in RLT and lysed with tissue lyser® (QIAGEN). Samples were passed through columns with washing steps and DNA incubation to purify RNAs. Elution was performed with RNAse free water, and RNAs concentration was evaluated with Nanodrop® 2000c (Thermo Scientific).
Genomic DNA was obtained with QiaAmp DNA Mini Kit (QiaGEN) following manufacturer’s instructions. Cells were lysed and passed through column to bind DNA, and after two washing steps genomic material was eluted in Elution Buffer. Genomic DNA concentration was measured using n Nanodrop® 2000c; and then stored at -20°C.
For qPCR, RNAs were reverse transcripted using High capacity reverse transcriptase (Invitrogen) and diluted at 50ng/µL in RNAse fre water. Then, qPCR was performed using Fast SYBR® Green Mix (Applied Biosystems) on 394 wells plate, results were acquired with Viia7 device (Life Technologies) and data were analyzed with Real-Time qPCR Studio (Life Technologies) using the 2-ΔΔCt method compared to CTL condition.
Paraformaldehyde (PFA) (4%) fixed muscles and ScAT were embedded in agarose 3% for 1 hour and sliced with Vibroslicer® 5100mz (Campden instruments) (300µm thick). Samples were permeabilized with Triton X100 (0,2% in PBS) before non-specific antigen saturation with BSA 3% and Goat serum in PBS. Tissue sections were incubated with Hamster anti mouse podoplanin, anti Sca1, anti CD45, anti-GFP primary antibodies for 24 hours at 4°C under constant agitation. Several washing with PBS were performed, and then sections were incubated with secondary antibodies (see Supplementary Informations) for 8 hours at RT. After several washes, nucleus were stained with DAPI and images were obtained using ZEISS LSM780 Confocal microscope (Plateforme I2MC) and analyzed with IMARIS® 8 (Bitplane).
Mouse muscle samples were cryopreserved in OCT frozen in liquid nitrogen–cooled isopentane. Samples were then sectioned at 10 μ on a cryostat and post fixed with 4% PFA for 15 min at RT. Slides were incubated with PBS containing Triton 0.5% (10 min, RT). They were incubated with 2% BSA (1h, RT) and overnight with a rabbit anti laminin antibody (1:200, Sigma Aldrich) (4°C, moist chamber) Slides were washed three times with PBS and incubated with FITC conjugated anti-rabbit secondary antibody (1:200, Invitrogen) (37°C, 45 min). Section were soaked for 5 min in DAPI solution and washed once with PBS before mounting with antifading mounting medium. Images were captured using Operetta High Content Imaging System (Perkin Elmer). The size and distribution of myofibers with central nuclei was calculated from laminin–DAPI staining on all fibers of the section and area determination were performed across the entire sections using an automated image processing algorithm (Harmony 4.5 software, Perkin Elmer).
Data are expressed as the mean ± s.e.m. Statistical analyses were performed using Student’s t-test (two-tailed paired or unpaired) or one-way ANOVA followed by the post hoc Dunnett’s and Tukey’s test with GraphPad Prism software (GraphPad Software). P values less than * p<0,05 ; ** p<0,01 and *** p<0,001 were statistically significant.